Newborns as a group are at increased risk for invasive bacterial infections and resulting sepsis. Although the majority of these infections in newborns are caused by gram-positive organisms, a variable but significant percentage of bacterial infections (about 20-40%) are due to gram-negative bacteria, particularly E. coli, Haemophilus influenzae, Klebsiella spp., and Enterobacter spp. In fact, it is the gram-negative infections that are, in some studies, associated with the highest mortality rate, which can be as high as about 40%. [Beck-Sague, C M et al., Pediatr Infect Dis J 13: 1110-116 (1994) and Stoll, B J et al., J Pediatr 129: 63-71 (1996)]
The mechanisms by which newborns are at increased risk for these bacterial infections are not currently understood. Although the neutrophil defense system is innate, there are indications that its function at birth is immature and suboptimal. Previous investigations of the activity of newborn neutrophils have demonstrated impaired adherence, chemotaxis, and phagocytosis. [Wright W C Jr. et al. Pediatrics 56: 579-584 (1975); Cairo M S, AJDC, 143:40-46 (1989); Schelonka R L et al., Sem. Perinatol., 22:2-14 (1998).] Impaired stimulus-induced adhesion and migration has been associated with decreased surface expression of L-selectin and the .beta..sub.2 -integrin Mac-1. [Dinauer, M C, in "Hematology of Infancy & Childhood," 5th ed., Nathan and Orkin, eds., Vol I, pp 889-967 (1998)] These findings may explain the difficulty in mobilizing neutrophils to sites of bacterial infection but do not explain the decreased phagocytic and bactericidal activity of the neutrophils of newborns.
Most studies of the microbicidal mechanism of newborn neutrophils have focused on the oxidative mechanism (i.e., the phagocyte oxidase/MPO/hydroxyl radical system), with conflicting data indicating either increased or decreased capacity of this oxygen-dependent mechanism in newborns. [Dinauer, supra, and Ambruso et al., Ped Res 18:1148-53 (1984).] Despite a growing literature on antibiotic proteins and peptides, little is known about the oxygen-independent microbicidal mechanisms of newborn neutrophils. A slightly decreased content of specific (secondary) granules in the neutrophils of newborns has been documented, with an associated modest (.ltoreq.2-fold) decrease in lysozyme and lactoferrin content relative to adult neutrophils. [Ambruso et al., supra.] However, the major elements of the oxygen-independent antimicrobial arsenal of neutrophil primary granules, including BPI and the defensin peptides, have not been assessed in neonates. Qing et al., Infect. Immun., 64:4638-4642 (1996), compared the lipopolysaccharide (LPS) binding of newborn neutrophils to that of adult neutrophils and reported that the newborn neutrophils have lower levels of membrane-associated 55-57 kDa and 25 kDa proteins capable of binding LPS. Although the missing proteins were not identified, the size and binding properties of the 55-57 kDa protein appeared to be similar to those of bactericidal/permeability-increasing protein (BPI) and the surface LPS receptor CD14.
The rising tide of antibiotic resistance has placed renewed emphasis on the development of agents to treat bacterial infection and its sequelae. Moreover, improved technology has led to increased survival rates for extremely ill full-term as well as premature neonates, which represent a growing population at high risk for bacterial infection. Although the replacement of neutrophils by granulocyte transfusion in newborns with sepsis has apparently been beneficial in some studies [Cairo et al., Pediatrics 74: 887-92 (1984)] this potential therapy has been complicated by difficulty in obtaining histocompatible neutrophils and by transfusion reactions.
BPI is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs or neutrophils), which are blood cells essential in the defense against invading microorganisms. Human BPI protein has been isolated from PMNs by acid extraction combined with either ion exchange chromatography [Elsbach, J. Bio. Chem., 254:11000 (1979)] or E. coli affinity chromatography [Weiss, et al., Blood, 69:652 (1987)]. BPI obtained in such a manner is referred to herein as natural BPI and has been shown to have potent bactericidal activity against a broad spectrum of gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 daltons (55 kD). The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding the protein have been reported in FIG. 1 of Gray et al., J. Bio. Chem., 264:9505 (1989), incorporated herein by reference. The Gray et al. amino acid sequence is set out in SEQ ID NO: 1 hereto. U.S. Pat. No. 5,198,541 discloses recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI.
BPI is a strongly cationic protein. The N-terminal half of BPI accounts for the high net positive charge; the C-terminal half of the molecule has a net charge of .+-.3. [Elsbach and Weiss (1981), supra.] A proteolytic N-terminal fragment of BPI having a molecular weight of about 25 kD possesses essentially all the anti-bacterial efficacy of the naturally-derived 55 kD human BPI holoprotein. [Ooi et al., J. Bio. Chem., 262: 14891-14894 (1987)]. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity against gram-negative organisms. [Ooi et al., J. Exp. Med., 174:649 (1991).] An N-terminal BPI fragment of approximately 23 kD, referred to as "rBPI.sub.23," has been produced by recombinant means and also retains anti-bacterial activity against gram-negative organisms. [Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).] An N-terminal analog of BPI, rBPI.sub.21, has been produced as described in Horwitz et al., Protein Expression Purification, 8:28-40 (1996).
The bactericidal effect of BPI was originally reported to be highly specific to gram-negative species, e.g., in Elsbach and Weiss, Inflammation: Basic Principles and Clinical Correlates, eds. Gallin et al., Chapter 30, Raven Press, Ltd. (1992). The precise mechanism by which BPI kills gram-negative bacteria is not yet completely elucidated, but it is believed that BPI must first bind to the surface of the bacteria through electrostatic and hydrophobic interactions between the cationic BPI protein and negatively charged sites on LPS. In susceptible gram-negative bacteria, BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death. [Elsbach and Weiss (1992), supra]. LPS has been referred to as "endotoxin" because of the potent inflammatory response that it stimulates, i.e., the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to lipid A, reported to be the most toxic and most biologically active component of LPS.
BPI protein products have a wide variety of beneficial activities. BPI protein products are bactericidal for gram-negative bacteria, as described in U.S. Pat. Nos. 5,198,541 and 5,523,288, both of which are incorporated herein by reference. International Publication No. WO 94/20130 (incorporated herein by reference) proposes methods for treating subjects suffering from an infection (e.g. gastrointestinal) with a species from the gram-negative bacterial genus Helicobacter with BPI protein products. BPI protein products also enhance the effectiveness of antibiotic therapy in gram-negative bacterial infections, as described in U.S. Pat. No. 5,523,288 and International Publication No. WO 95/08344 (PCT/US94/11255), which are incorporated herein by reference. BPI protein products are also bactericidal for gram-positive bacteria and mycoplasma, and enhance the effectiveness of antibiotics in gram-positive bacterial infections, as described in U.S. Pat. Nos. 5,578,572 and 5,783,561 and International Publication No. WO 95/19180 (PCT/US95/00656), which are incorporated herein by reference. BPI protein products exhibit anti-fungal activity, and enhance the activity of other anti-fungal agents, as described in U.S. Pat. No. 5,627,153 and International Publication No. WO 95/19179 (PCT/US95/00498), and further as described for anti-fungal peptides in U.S. Pat. No. 5,858,974, which is in turn a continuation-in-part of U.S. application Ser. No. 08/504,841 filed Jul. 20, 1994 and corresponding International Publication Nos. WO 96/08509 (PCT/US95/09262) and WO 97/04008 (PCT/US96/03845), all of which are incorporated herein by reference. BPI protein products exhibit anti-protozoan activity, as described in U.S. Pat. No. 5,646,114 and International Publication No. WO 96/01647 (PCT/US95/08624), which are incorporated herein by reference. BPI protein products exhibit anti-chlamydial activity, as described in co-owned, co-pending U.S. application Ser. No. 08/694,843 filed Aug. 9, 1996 and WO 98/06415 (PCT/US97/13810), which are incorporated herein by reference. Finally, BPI protein products exhibit anti-mycobacterial activity, as described in co-owned, co-pending U.S. application Ser. No. 08/626,646 filed Apr. 1, 1996, which is in turn a continuation of U.S. application Ser. No. 08/285,803 filed Aug. 14, 1994, which is in turn a continuation-in-part of U.S. application Ser. No. 08/031,145 filed Mar. 12, 1993 and corresponding International Publication No. WO94/20129 (PCT/US94/02463), all of which are incorporated herein by reference.
The effects of BPI protein products in humans with endotoxin in circulation, including effects on TNF, IL-6 and endotoxin are described in U.S. Pat. No. 5,643,875, which is incorporated herein by reference.
BPI protein products are also useful for treatment of specific disease conditions, such as meningococcemia in humans (as described in co-owned, co-pending U.S. application Ser. No. 08/644,287 filed May 10, 1996 and continuation No. 08/927,437 filed Sep. 10, 1997 and International Publication No. WO97/42966 (PCT/US97/08016), all of which are incorporated herein by reference), hemorrhagic trauma in humans, (as described in U.S. Pat. No. 5,756,464, U.S. application Ser. No. 08/862,785 filed May 23, 1997 and corresponding International Publication No. WO 97/44056 (PCT/US97/08941), all of which are incorporated herein by reference), burn injury (as described in U.S. Pat. No. 5,494,896, which is incorporated herein by reference), ischemia/reperfusion injury (as described in U.S. Pat. No. 5,578,568, incorporated herein by reference), and liver resection (as described in co-owned, co-pending U.S. application Ser. No. 08/582,230 filed Jan. 3, 1996, which is in turn a continuation of U.S. application Ser. No. 08/318,357 filed Oct. 5, 1994, which is in turn a continuation-in-part of U.S. application Ser. No. 08/132,510 filed Oct. 5, 1993, and corresponding International Publication No. WO 95/10297 (PCT/US94/11404), all of which are incorporated herein by reference).
BPI protein products also neutralize the anti-coagulant activity of exogenous heparin, as described in U.S. Pat. No. 5,348,942, incorporated herein by reference, and are useful for treating chronic inflammatory diseases such as rheumatoid and reactive arthritis and for inhibiting angiogenesis and for treating angiogenesis-associated disorders including malignant tumors, ocular retinopathy and endometriosis, as described in U.S. Pat. Nos. 5,639,727, 5,807,818 and 5,837,678 and International Publication No. WO 94/20128 (PCT/US94/02401), all of which are incorporated herein by reference.
BPI protein products are also useful in antithrombotic methods, as described in U.S. Pat. No. 5,741,779 and U.S. application Ser. No. 09/063,465 filed Apr. 20, 1998 and corresponding WO 97/42967 (PCT/US7/08017), all of which are incorporated herein by reference.